Global genome initiatives have generated enormous amounts of information, spurned new technologies and catalyzed the emergence of a new type of biology which attempts to build biological knowledge from systematic and quantitative analysis of the molecules that constitute a cell or tissue. Expectations for applying comprehensive and quantitative analyses of genes and proteins to cancer biology are high. Comparative profiling of mRNA & proteins expressed in "normal" and cancer cells and tissues is expected to discover new markers for diagnosis of type, stage and stratification of cancer, indicators to assess success of treatment and emergence of drug resistance, and lead to an understanding of the molecular basis of cancer and thus new therapeutic targets. We propose to develop a novel technology for quantitative, sensitive and comprehensive analysis of protein expression profiles in human cells and tissues. It is based on quantitative, automated mass spectrometry of complex peptide mixtures and consists of 2 phases. First is generation of a reference peptide database of a given cell type in which each peptide is annotated with characteristics that uniquely identify it in the database. Second is correlation of mass spectrometric data obtained from clinical or research samples with the peptide database to identify and quantify protein components of the cell type. The technology is: 1) automatable, 2) compatible with analysis of essentially any protein, 3) robust and easy to standardize and 4) amenable to high throughput. This technology will provide a general/robust technical basis for comprehensive and quantitative analysis of protein profiles of normal and cancer cells and tissues that will be widely accessible. The new technology will be validated and applied to prostate cancer research. Specifically, we will correlate the data obtained from the quantitative protein profiling technology developed in this program with gene expression data obtained via cDNA arrays and Lynx Therapeutic's massively parallel signature sequencing (MPSS) procedure to identify biologic determinants that differentiate indolent and aggressive prostate cancer and to determine the molecular basis of androgen dependent cell growth of prostate cells. The application satisfies the request for innovative technology development in 3 ways: First, the technology explores a new approach to identification and quantification of the comprehensive spectrum of proteins expressed in human cells/tissues; Second, the annotated peptide database (APD) for prostate tissue will be made available publicly and serve as a necessary reference for quantitative protein expression profiling in laboratories not currently specialized in proteomics and Third, research will be conducted as an academic/industrial partnership assuring rapid commercial accessibility of reagents/instrumentation supporting the technology. We anticipate the proposed pro ram will contribute significant towards fulfilling expectations for cancer research raised by the genomics revolution.